Small molecule PIKfyve inhibitors as cancer therapeutics: Translational promises and limitations.

Through synthesis of two rare phosphoinositides, PtdIns(3,5)P2 and PtdIns5P, the ubiquitously expressed phosphoinositide kinase PIKfyve is implicated in pleiotropic cellular functions. Small molecules specifically inhibiting PIKfyve activity cause cytoplasmic vacuolation in all dividing cells in culture yet trigger non-apoptotic death through excessive vacuolation only in cancer cells. Intriguingly, cancer cell toxicity appears to be inhibitor-specific suggesting that additional targets beyond PIKfyve are affected. One PIKfyve inhibitor - apilimod - is already in clinical trials for treatment of B-cell malignancies. However, apilimod is inactivated in cultured cells and exhibits unexpectedly low plasma levels in patients treated with maximum oral dosage. Thus, the potential widespread use of PIKfyve inhibitors as cancer therapeutics requires progress on multiple fronts: (i) advances in methods for isolating relevant cancer cells from individual patients; (ii) delineation of the molecular mechanisms potentiating the vacuolation induced by PIKfyve inhibitors in sensitive cancer cells; (iii) design of PIKfyve inhibitors with favorable pharmacokinetics; and (iv) development of effective drug combinations.

PIKfyve inhibitor cytotoxicity requires AKT suppression and excessive cytoplasmic vacuolation.

PIKfyve phosphoinositide kinase produces PtdIns(3,5)P2 and PtdIns5P and governs a myriad of cellular processes including cytoskeleton rearrangements and cell proliferation. The latter entails rigorous investigation since the cytotoxicity of PIKfyve inhibition is a potential therapeutic modality for cancer. Here we report the effects of two PIKfyve-specific inhibitors on the attachment/spreading and viability of mouse embryonic fibroblasts (MEFs) and C2C12 myoblasts. Importantly, 18-h treatment of adherent cells with YM201636 (800 nM) and apilimod (20 nM) in serum-containing culture media did not affect cell viability despite the presence of multiple cytoplasmic vacuoles, a hallmark of PIKfyve inhibition. Strikingly, at the same dose and duration the inhibitors caused excessive cytoplasmic vacuolation, initial suppression of cell attachment/spreading and subsequent marked detachment/death in serum-deprived cells. The remaining adherent cells under serum-deprived conditions had smaller surface area, lacked vinculin/actin-positive focal adhesions and displayed vacuoles occupying the entire cytoplasm. Serum or growth factors protected against PIKfyve inhibitor cytotoxicity. This protection required Akt activation evidenced by the abrogated beneficial effect of serum upon treatment with the clinically-relevant Akt inhibitor MK-2206. Moreover, Akt inhibition triggered cell detachment/death even in serum-fed adherent MEFs treated with apilimod. Intriguingly, BafilomycinA1 (H+-vacuolar ATPase inhibitor), which prevents the cytoplasmic vacuolation under PIKfyve perturbations, rescued all defects in attaching/spreading as well as in adherent cells under serum-starved or serum-fed conditions, respectively. Together, the results indicate that the cytotoxicity of PIKfyve inhibitors in MEFs and C2C12 myoblasts requires Akt suppression and excessive cytoplasmic vacuolation.

GAPDH binds Akt to facilitate cargo transport in the early secretory pathway.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes numerous post-translational modifications, which impart new function and influence intracellular location. For example, atypical PKC ι/λ phosphorylates GAPDH that locates to vesicular tubular clusters and is required for retrograde membrane trafficking in the early secretory pathway. GAPDH is also required in the endocytic pathway; substitution of Pro234 to Ser (Pro234Ser) rendered CHO cells defective in endocytosis. To determine if GAPDH (Pro234Ser) could inhibit endoplasmic reticulum to Golgi trafficking, we introduced the recombinant mutant enzyme into several biochemical and morphological transport assays. The mutant protein efficiently blocked vesicular stomatitis virus-G protein transport. Because GAPDH binds to microtubules (MTs), we evaluated MT binding and MT intracellular distribution in the presence of the mutant. Although these properties were not changed relative to wild-type, GAPDH (Pro234Ser) altered Golgi complex morphology. We determined that the GAPDH point mutation disrupted association between the enzyme and the serine/threonine kinase Akt. Interestingly Rab1, which functions in anterograde-directed trafficking, stimulates GAPDH-Akt association with membranes in a quantitative binding assay. In contrast, Rab2 does not stimulate GAPDH-Akt membrane binding but instead recruits GAPDH-aPKC. We propose a mechanism whereby the association of GAPDH with Akt or with aPKC serves as a switch to discriminate between anterograde directed cargo and recycling cargo retrieved back to the ER, respectively.

Plentiful PtdIns5P from scanty PtdIns(3,5)P2 or from ample PtdIns? PIKfyve-dependent models: Evidence and speculation (response to: DOI 10.1002/bies.201300012).

Recently, we have presented data supporting the notion that PIKfyve not only produces the majority of constitutive phosphatidylinositol 5-phosphate (PtdIns5P) in mammalian cells but that it does so through direct synthesis from PtdIns. Another group, albeit obtaining similar data, suggests an alternative pathway whereby the low-abundance PtdIns(3,5)P2 undergoes hydrolysis by unidentified 3-phosphatases, thereby serving as a precursor for most of PtdIns5P. Here, we review the experimental evidence supporting constitutive synthesis of PtdIns5P from PtdIns by PIKfyve. We further emphasize that the experiments presented in support of the alternative pathway are also compatible with a direct mechanism for PIKfyve-catalyzed synthesis of PtdIns5P. While agreeing with the authors that constitutive PtdIns5P could theoretically be produced from PtdIns(3,5)P2 by 3-dephosphorylation, we argue that until direct evidence for such an alternative pathway is obtained, we should adhere to the existing experimental evidence and quantitative considerations, which favor direct PIKfyve-catalyzed synthesis for most constitutive PtdIns5P.

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells.

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34-deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux.

Host PI(3,5)P2 activity is required for Plasmodium berghei growth during liver stage infection.

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5-kinase (PIKfyve) that converts phosphatidylinositol 3-phosphate [PI(3)P] into phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2 ] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non-pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P2 effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P2 -dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.

Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency.

The two evolutionarily conserved mammalian lipid kinases Vps34 and PIKfyve are involved in an important physiological relationship, whereby the former produces phosphatidylinositol (PtdIns) 3P that is used as a substrate for PtdIns(3,5)P2 synthesis by the latter. Reduced production of PtdIns(3,5)P2 in proliferating mammalian cells is phenotypically manifested by the formation of multiple translucent cytoplasmic vacuoles, readily rescued upon exogenous delivery of PtdIns(3,5)P2 or overproduction of PIKfyve. Although the aberrant vacuolation phenomenon has been frequently used as a sensitive functional measure of localized PtdIns(3,5)P2 reduction, cellular factors governing the appearance of cytoplasmic vacuoles under PtdIns3P-PtdIns(3,5)P2 loss remain elusive. To gain further mechanistic insight about the vacuolation process following PtdIns(3,5)P2 reduction, in this study we sought for cellular mechanisms required for manifestation of the aberrant endomembrane vacuoles triggered by PIKfyve or Vps34 dysfunction. The latter was achieved by various means such as pharmacological inhibition, gene disruption, or dominant-interference in several proliferating mammalian cell types. We report here that inhibition of V-ATPase with bafilomycin A1 as well as inactivation of the GTP-GDP cycle of Rab5a GTPase phenotypically rescued or completely precluded the cytoplasmic vacuolization despite the continued presence of inactivated PIKfyve or Vps34. Bafilomycin A1 also restored the aberrant EEA1-positive endosomes, enlarged upon short PIKfyve inhibition with YM201636. Together, our work identifies for the first time that factors such as active V-ATPase or functional Rab5a cycle are acting coincidentally with the PtdIns(3,5)P2 reduction in triggering formation of aberrant cytoplasmic vacuoles under PIKfyve or Vps34 dysfunction.

The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation.

The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders.

Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.

The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.

cdc-like/dual-specificity tyrosine phosphorylation-regulated kinases inhibitor leucettine L41 induces mTOR-dependent autophagy: implication for Alzheimer's disease.

Leucettines, a family of pharmacological inhibitors of dual-specificity tyrosine phosphorylation regulated kinases and cdc-like kinases (CLKs), are currently under investigation for their potential therapeutic application to Down syndrome and Alzheimer's disease. We here report that leucettine L41 triggers bona fide autophagy in osteosarcoma U-2 OS cells and immortalized mouse hippocampal HT22 cells, characterized by microtubule-associated protein light chain 3 membrane translocation and foci formation. Leucettine L41-triggered autophagy requires the Unc-51-like kinase and is sensitive to the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and 3-methyladenine, suggesting that it acts through the mammalian target of rapamycin (mTOR)/PI3K-dependent pathway. Leucettine L41 does not act by modifying the autophagic flux of vesicles. Leucettine L41-induced autophagy correlates best with inhibition of CLKs. Leucettine L41 modestly inhibited phosphatidylinositol-3-phosphate 5-kinase, FYVE domain-containing activity as tested both in vitro and in vivo, which may also contribute to autophagy induction. Altogether these results demonstrate that leucettines can activate the autophagic mTOR/PI3K pathway, a characteristic that may turn advantageous in the context of Alzheimer's disease treatment.

Muscle-specific Pikfyve gene disruption causes glucose intolerance, insulin resistance, adiposity, and hyperinsulinemia but not muscle fiber-type switching.

The evolutionarily conserved kinase PIKfyve that synthesizes PtdIns5P and PtdIns(3,5)P2 has been implicated in insulin-regulated GLUT4 translocation/glucose entry in 3T3-L1 adipocytes. To decipher PIKfyve's role in muscle and systemic glucose metabolism, here we have developed a novel mouse model with Pikfyve gene disruption in striated muscle (MPIfKO). These mice exhibited systemic glucose intolerance and insulin resistance at an early age but had unaltered muscle mass or proportion of slow/fast-twitch muscle fibers. Insulin stimulation of in vivo or ex vivo glucose uptake and GLUT4 surface translocation was severely blunted in skeletal muscle. These changes were associated with premature attenuation of Akt phosphorylation in response to in vivo insulin, as tested in young mice. Starting at 10-11 wk of age, MPIfKO mice progressively accumulated greater body weight and fat mass. Despite increased adiposity, serum free fatty acid and triglyceride levels were normal until adulthood. Together with the undetectable lipid accumulation in liver, these data suggest that lipotoxicity and muscle fiber switching do not contribute to muscle insulin resistance in MPIfKO mice. Furthermore, the 80% increase in total fat mass resulted from increased fat cell size rather than altered fat cell number. The observed profound hyperinsulinemia combined with the documented increases in constitutive Akt activation, in vivo glucose uptake, and gene expression of key enzymes for fatty acid biosynthesis in MPIfKO fat tissue suggest that the latter is being sensitized for de novo lipid anabolism. Our data provide the first in vivo evidence that PIKfyve is essential for systemic glucose homeostasis and insulin-regulated glucose uptake/GLUT4 translocation in skeletal muscle.

The PIKfyve-ArPIKfyve-Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines.

The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve-ArPIKfyve-Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P-PtdIns(3,5)P2 synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P-PtdIns(3,5)P2 conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P2 in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or indirectly the homeostasis of these lipids in TNBC. Together, our results uncover an unexpected role for Sac3 phosphatase in TNBC cell proliferation. Database analyses, discussed herein, reinforce the involvement of Sac3 in breast cancer pathogenesis.

PIKfyve and its Lipid products in health and in sickness.

PIKfyve, a phosphoinositide 5-kinase synthesizing PtdIns(3,5)P2 and PtdIns5P in a cellular context, belongs to an evolutionarily ancient gene family of PtdIns(3,5)P2-synthesizing enzymes that, except for plants, are products of a single-copy gene across species. In the dozen years after its discovery, enormous progress has been made in characterizing the numerous PIKfyve cellular functions and the regulatory mechanisms that govern these functions. It became clear that PIKfyve does not act alone but, rather, it engages the scaffolding regulator ArPIKfyve and the phosphatase Sac3 to make a multiprotein "PAS" complex, so called for the first letters of the protein names. This complex relays antagonistic signals, one for synthesis, another for turnover of PtdIns(3,5)P2, whose dysregulated coordination is linked to several human diseases. The physiological significance for each protein in the PAS complex is underscored by the early lethality of the mouse models with disruption in any of the three genes. This chapter summarizes our current knowledge of the diverse and complex functionality of PIKfyve and PtdIns(3,5)P2/PtdIns5P products with particular highlights on recent discoveries of inherited or somatic mutations in PIKfyve and Sac3 linked to human disorders.

PtdIns5P: news and views of its appearance, disappearance and deeds.

Accumulated evidence indicates that PtdIns5P, one of the seven phosphoinositides, found now to be constitutively present in yeast, plants and metazoa, serves as a signaling molecule to modulate pleiotropic cellular functions in both the nucleus and the cytoplasm. The enzymatic routes in biogenesis of basal PtdIns5P have remained incompletely understood. The role for candidate kinase PIKfyve that is principally involved in PtdIns(3,5)P2 production, has been questioned. In this review article we scrutinize the past obstacles that prevented the definitive implication of PIKfyve in PtdIns5P biosynthesis from PtdIns and focus on the recent pharmacological and genetic advancements that now make this conclusion well supported. We further summarize our current knowledge of the diverse stimuli modulating PtdIns5P levels, binding partners and regulated cellular process, with particular reference to the available mechanistic insights for the relevant signaling pathways.

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636.

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice.

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P(2) and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve(KO/KO) mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve(flox/flox) embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve(WT/KO) mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve(WT/KO) mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50-55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P(2) and PtdIns5P selectively decreased, but this reduction (35-40%) was 10-15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve(WT/KO) mice is able to support the demands for PtdIns(3,5)P(2)/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.

ArPIKfyve regulates Sac3 protein abundance and turnover: disruption of the mechanism by Sac3I41T mutation causing Charcot-Marie-Tooth 4J disorder.

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P(2)) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P(2) production. Whether the ArPIKfyve-Sac3 subcomplex functions outside the PIKfyve context is unknown. Here we show that stable or transient expression of ArPIKfyve(WT) in mammalian cells elevates steady-state protein levels and the PtdIns(3,5)P(2)-hydrolyzing activity of Sac3, whereas knockdown of ArPIKfyve has the opposite effect. These manipulations do not alter the Sac3 mRNA levels, suggesting that ArPIKfyve might control Sac3 protein degradation. Inhibition of protein synthesis in COS cells by cycloheximide reveals remarkably rapid turnover of expressed Sac3(WT) (t((1/2)) = 18.8 min), resulting from a proteasome-dependent clearance as evidenced by the extended Sac3(WT) half-life upon inhibiting proteasome activity. Coexpression of ArPIKfyve(WT), but not the N- or C-terminal halves, prolongs the Sac3(WT) half-life consistent with enhanced Sac3 protein stability through association with full-length ArPIKfyve. We further demonstrate that mutant Sac3, harboring the pathogenic Ile-to-Thr substitution at position 41 found in patients with CMT4J disorder, is similar to Sac3(WT) with regard to PtdIns(3,5)P(2)-hydrolyzing activity, association with ArPIKfyve, or rapid proteasome-dependent clearance. Remarkably, however, neither is the steady-state Sac3(I41T) elevated nor is the Sac3(I41T) half-life extended by coexpressed ArPIKfyve(WT), indicating that unlike with Sac3(WT), ArPIKfyve fails to prevent Sac3(I41T) rapid loss. Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and suggest that a failure of this mechanism could be the primary molecular defect in the pathogenesis of CMT4J.

PIKfyve-ArPIKfyve-Sac3 core complex: contact sites and their consequence for Sac3 phosphatase activity and endocytic membrane homeostasis.

The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P(2) biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P(2) hydrolyzing activity within the PAS complex. Herein, we address the issue of Sac3 functionality by a combination of biochemical and morphological assays in triple-transfected COS cells using a battery of truncated or point mutants of the three proteins. We identified the Cpn60_TCP1 domain of PIKfyve as a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Neither Sac3 nor PIKfyve enzymatic activities affected the PAS complex formation or stability. Using the well established formation of aberrant cell vacuoles as a sensitive functional measure of localized PtdIns(3,5)P(2) reduction, we observed a mitigated vacuolar phenotype by kinase-deficient PIKfyve(K1831E) if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P(2). In contrast, PIKfyve(K1831E), which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyve(WT)-Sac3(WT) but minimal defects when coexpressed with ArPIKfyve(WT) and phosphatase-deficient Sac3(D488A). These data indicate that Sac3 assembled in the PAS regulatory core complex is an active PtdIns(3,5)P(2) phosphatase. Based on these and other data, presented herein, we propose a model of domain interactions within the PAS core and their role in regulating the enzymatic activities.

YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes.

Silencing of PIKfyve, the sole enzyme for PtdIns(3,5)P(2) biosynthesis that controls proper endosome dynamics, inhibits retroviral replication. A novel PIKfyve-specific inhibitor YM201636 disrupts retroviral budding at 800 nM, suggesting its potential use as an antiretroviral therapeutic. Because PIKfyve is also required for optimal insulin activation of GLUT4 surface translocation and glucose influx, we tested the outcome of YM201636 application on insulin responsiveness in 3T3L1 adipocytes. YM201636 almost completely inhibited basal and insulin-activated 2-deoxyglucose uptake at doses as low as 160 nM, with IC(50)=54+/-4 nM for the net insulin response. Insulin-induced GLUT4 translocation was partially inhibited at substantially higher doses, comparable to those required for inhibition of insulin-induced phosphorylation of Akt/PKB. In addition to PIKfyve, YM201636 also completely inhibited insulin-dependent activation of class IA PI 3-kinase. We suggest that apart from PIKfyve, there are at least two additional targets for YM201636 in the context of insulin signaling to GLUT4 and glucose uptake: the insulin-activated class IA PI 3-kinase and a here-unidentified high-affinity target responsible for the greater inhibition of glucose entry vs. GLUT4 translocation. The profound inhibition of the net insulin effect on glucose influx at YM201636 doses markedly lower than those required for efficient retroviral budding disruption warns of severe perturbations in glucose homeostasis associated with potential YM201636 use in antiretroviral therapy.

Phosphatidylinositol 3-phosphate [PtdIns3P] is generated at the plasma membrane by an inositol polyphosphate 5-phosphatase: endogenous PtdIns3P can promote GLUT4 translocation to the plasma membrane.

Exogenous delivery of carrier-linked phosphatidylinositol 3-phosphate [PtdIns(3)P] to adipocytes promotes the trafficking, but not the insertion, of the glucose transporter GLUT4 into the plasma membrane. However, it is yet to be demonstrated if endogenous PtdIns(3)P regulates GLUT4 trafficking and, in addition, the metabolic pathways mediating plasma membrane PtdIns(3)P synthesis are uncharacterized. In unstimulated 3T3-L1 adipocytes, conditions under which PtdIns(3,4,5)P3 was not synthesized, ectopic expression of wild-type, but not catalytically inactive 72-kDa inositol polyphosphate 5-phosphatase (72-5ptase), generated PtdIns(3)P at the plasma membrane. Immunoprecipitated 72-5ptase from adipocytes hydrolyzed PtdIns(3,5)P2, forming PtdIns(3)P. Overexpression of the 72-5ptase was used to functionally dissect the role of endogenous PtdIns(3)P in GLUT4 translocation and/or plasma membrane insertion. In unstimulated adipocytes wild type, but not catalytically inactive, 72-5ptase, promoted GLUT4 translocation and insertion into the plasma membrane but not glucose uptake. Overexpression of FLAG-2xFYVE/Hrs, which binds and sequesters PtdIns(3)P, blocked 72-5ptase-induced GLUT4 translocation. Actin monomer binding, using latrunculin A treatment, also blocked 72-5ptase-stimulated GLUT4 translocation. 72-5ptase expression promoted GLUT4 trafficking via a Rab11-dependent pathway but not by Rab5-mediated endocytosis. Therefore, endogenous PtdIns(3)P at the plasma membrane promotes GLUT4 translocation.